Project Summary The degradation of misfolded proteins in the endoplasmic reticulum (ER) by the ER-associated degradation (ERAD) pathway prevents potentially toxic proteins from entering the secretory pathway. ERAD, however, cannot clear all proteins from the ER. For example, some proteins, such as aggregation-prone proteins, large polymers and fibrillar proteins, are resistant to degradation by ERAD and must be disposed of by alternate disposal pathways. As aggregation prone proteins have been to linked to neurodegenerative diseases, understanding how these alternate disposal pathways function is of medical importance. ER autophagy (ER-phagy) is a disposal pathway that degrades ER domains and aggregation-prone proteins. How specific domains, on the continuous network of the ER, are targeted for degradation is unknown. We have found that a non-canonical form of the COPII coat, that contains SEC24C-SEC23, works with receptors on the ER to target domains for autophagy. ER-phagy sites (ERPHS) on the ER are distinct from the ER exit sites where secretory cargo is loaded into canonical COPII coated vesicles that traffic to the Golgi. Our findings suggest that ER structure may be important for the formation of ERPHS. Additionally, mutations in several ER shaping proteins, associated with hereditary spastic paraplegias (HSP), lead to defects in ER-phagy. These findings suggest a link between ER-phagy, the formation of the ERPHS and HSP. In this proposal I describe several aims that are designed to address the role that ER structure plays in the formation of ERPHS and the link between ERPHS formation and HSP. Specifically, we will perform live cell imaging and mass spectroscopy experiments to characterize the ERPHS and their cargo. Misfolded proteins, known to be degraded by ER-phagy, will be analyzed. To date six ER autophagy receptors have been identified. Our studies will address when SEC24C interacts with the autophagy machinery and which of the six known receptors interact with SEC24C. Our biochemical studies may lead to the identification of new proteins that interact with SEC24C during ER autophagy. Autophagy reporters, imaging analysis and biochemical studies will be used to address the role that ER organization and ER shaping proteins play in ER-phagy and ERPHS formation. The proteins we will analyze in Aim 2 and Aim 3 are associated with HSP and HSP-like neuropathies. In total, these studies will shed light on the link between ER structure, ERPHS formation and HSP.